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In vitro phosphorylation of von Willebrand factor by FAM20c enhances its ability to support platelet adhesion.

ABSTRACT: Essentials Platelet adhesion to von Willebrand factor (VWF) is critical for hemostasis and thrombosis. Whether VWF can undergo phosphorylation is unknown. Family with sequence similarity 20 kinase phosphorylates VWF A2 domain at S1517 and S1613. Phosphorylation of VWF and VWF A1A2A3 domain at S1613 enhances platelet adhesion. SUMMARY: Background von Willebrand factor (VWF) mediates platelet adhesion and contributes to hemostasis at sites of vascular injury as well as to arterial thrombosis. The A1A2A3 domains of VWF contain important sites that differentially participate in supporting platelet adhesion. FAM20c (family with sequence similarity 20, member C) has emerged as a serine/threonine kinase, which phosphorylates extracellular proteins containing the S-X-E/pS motifs that are also found within the VWF A domains. This is of interest because we and others have shown that structural modifications within these A domains influence the ability of VWF to support platelet adhesion. Objective We assessed if VWF A domains can be phosphorylated and the functional consequence of phosphorylated VWF. Results Here, we show that FAM20c phosphorylated purified plasma VWF, VWF A1A2A3 protein, isolated A2 domain, but not A1 and A3 domain proteins, in vitro. FAM20c phosphorylated the isolated A2 domain at S1517 and S1613 within the S-X-E recognition motif, with S1613 being the major phosphorylation site. Mass spectrometry analysis of purified plasma VWF from healthy donors revealed several phosphorylation sites, including the S1613 in the A2 domain. VWF A1A2A3 domain protein phosphorylated at S1613 promoted stable platelet adhesion and microthrombi at high shear stress. Lastly, under high shear stress VWF treated with FAM20c and ATP robustly supported platelet adhesion, compared to VWF treated with FAM20c in the absence of ATP. Conclusion These outcomes indicate that VWF can be phosphorylated by FAM20c in vitro, and this novel post-translational modification enhances the adhesiveness of VWF to platelets.

SUBMITTER: Da Q

PROVIDER: S-EPMC6545126 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Essentials The role of von Willebrand factor (VWF) domains in regulating platelet adhesion was studied in vivo. Multimeric VWF with spacers at the N- and C-terminus of VWF-A1 were systematically tested. N-terminal modified VWF avidly bound platelet GpIbα, causing VWD Type2B like phenotype in mice. Novel anti-D'D3 mAbs suggest that changes at the D'D3-A1 interface may be biologically relevant.SummaryBackground Previous ex vivo studies using truncated VWF (von Willebrand factor) suggest that domain-level molecular architecture may control platelet-GpIbα binding function. Objective We determined if this is the case with multimeric VWF in vivo. Methods Full-length human VWF ('hV') was modified with a 22-amino acid mucinous stretch at either the N-terminus of VWF-A1 to create 'hNV' or C-terminus to yield 'hCV'. This extends the physical distance between VWF-A1 and the adjacent domains by ~6 nm. Similar mucin inserts were also introduced into a human-murine chimera ('h[mA1]V') where murine-A1 replaced human-A1 in hV. This yielded 'h[mA1]NV' and 'h[mA1]CV', with N- and C-terminal inserts. The constructs were tested ex vivo and in vivo. Results Mucin insertion at the N-terminus, but not C-terminus, in both types of constructs resulted in >50-fold increase in binding to immobilized GpIbα. N-terminal insertion also resulted in greater shear-induced platelet activation, more thrombus formation on collagen, enhanced platelet accumulation and slower platelet translocation on immobilized VWF in microfluidics assays. Hydrodynamic injection-based expression of h[mA1]NV, but not h[mA1]V or h[mA1]CV, in VWF-/- mice caused profound thrombocytopenia, reduced plasma VWF concentrations, lower multimer distribution, and incessant tail bleeding that is reminiscent of von Willebrand disease type 2B. Platelet plugs were noted in the portal veins and hepatic arteries. An anti-D'D3 mAb DD3.3 that displays enhanced binding to VWF containing the N-terminal mucin insert also exhibited increased binding to wild-type VWF under shear and upon ristocetin addition. Conclusion Conformation changes at the VWF D'D3-A1 interface may be a key regulator of thrombosis in vivo. Structural features at the A1-A2 interface are likely of less significance.

Project description:BACKGROUND:Complete mechanistic understanding of impaired microvascular reflow after myocardial infarction will likely lead to new therapies for reducing infarct size. Myocardial contrast echocardiography perfusion imaging and molecular imaging were used to evaluate the contribution of microvascular endothelial-associated VWF (von Willebrand factor) and platelet adhesion to microvascular no-reflow. METHODS AND RESULTS:Myocardial infarction was produced by transient LAD ligation in WT (wild type) mice, WT mice treated with the VWF proteolytic enzyme ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type 1 motif, member 13), and ADAMTS13-deficient (ADAMTS13-/-) mice. Myocardial contrast echocardiography perfusion imaging and molecular imaging of VWF and platelet GP (glycoprotein) Ib? were performed 30 minutes after ischemia-reperfusion. Infarct size was measured at 3 days. Mortality during ischemia-reperfusion incrementally increased in WT+ADAMTS13, WT, and ADAMTS13-/- mice (14%, 43%, and 63%, respectively; P<0.05). For WT mice, molecular imaging signal for platelets and VWF in the postischemic risk area was 4- to 5-fold higher ( P<0.05) compared with both the remote nonischemic regions or to sham-treated mice. Signal enhancement in the risk area was completely abolished by ADAMTS13 treatment for both platelets (12.8±3.3 versus -1.0±4.4 IU; P<0.05) and VWF (13.9±4.0 versus -1.0±3.0 IU; P<0.05). ADAMTS13-/- compared with WT mice had 2- to 3-fold higher risk area signal for platelets (33.1±8.5 IU) and VWF (30.9±1.9 IU). Microvascular reflow in the risk area incrementally decreased for WT+ADAMTS13, WT, and ADAMTS13-/- mice ( P<0.05), whereas infarct size incrementally increased ( P<0.05). CONCLUSIONS:Mechanistic information on microvascular no-reflow is possible by combining perfusion and molecular imaging. In reperfused myocardial infarction, excess endothelial-associated VWF and secondary platelet adhesion in the risk area microcirculation contribute to impaired reflow and are modifiable.

Project description:Essentials von Willebrand factor (VWF) is synthesized in endothelial cells and platelet precursors. Type 3 patients with Pro2808Leufs*24 have lower bleeding scores than other type 3s. The Pro2808Leufs*24 variant was examined in patient platelets and endothelial cells. Type 3s with this variant contain releaseable VWF, possibly reducing bleeding. SUMMARY:Background A novel variant, p.Pro2808Leufs*24, in the von Willebrand factor (VWF) gene was previously identified in the Canadian von Willebrand disease (VWD) patient population. Clinical observations of type 3 VWD patients with this variant indicate a milder bleeding phenotype compared with other type 3 patients. Objective To assess the effect of the Pro2808Leufs*24 variant on the molecular pathogenesis of VWD and correlate this with the phenotype observed in patients. Patients/Methods Phenotypic data from individuals in the Canadian type 3 VWD study were analyzed. VWF expression in platelets and plasma was assessed via immunoblotting. Cellular expression of VWF in platelets and blood outgrowth endothelial cells (BOEC) was examined via immunofluorescence microscopy and biochemical analysis in a type 3 index case and family member with Pro2808Leufs*24. Results Twenty-six individuals with the Pro2808Leufs*24 variant (16 type 3 VWD homozygous or compound heterozygous and 10 heterozygous family members) were studied. Bleeding scores were lower in type 3 patients with Pro2808Leufs*24 compared with type 3 patients with other variants, confirming a milder bleeding phenotype. Immunoblotting of platelet lysates detected VWF in the platelets of type 3 patients with Pro2808Leufs*24. Examination of an index case detected VWF within platelets via immunofluorescence microscopy, and in vitro experiments showed that this VWF was released upon platelet activation. Patient BOECs showed decreased VWF synthesis and secretion, although some VWF-containing granules were observed. Conclusion Type 3 VWD patients with the Pro2808Leufs*24 have bioavailable platelet-derived VWF that may produce a milder bleeding phenotype than other type 3s.

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In vitro phosphorylation of von Willebrand factor by FAM20c enhances its ability to support platelet adhesion.

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